System and Method for Measuring Particles in a Sample Stream of a Flow Cytometer Using Low-Power Laser Source

ABSTRACT

A system and method for analyzing a particle in a sample stream of a flow cytometer or the like. The system has a light source, such as a laser pointer module, for generating a low powered light beam and a fluidics apparatus which is configured to transport particles in the sample stream at substantially low velocity through the light beam for interrogation. Detectors, such as photomultiplier tubes, are configured to detect optical signals generated in response to the light beam impinging the particles. Signal conditioning circuitry is connected to each of the detectors to condition each detector output into electronic signals for processing and is designed to have a limited frequency response to filter high frequency noise from the detector output signals.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of and now-allowed U.S. patentapplication Ser. No. 12/903,003, filed Oct. 12, 2010. U.S. patentapplication Ser. No. 12/903,003 is a continuation of U.S. patentapplication Ser. No. 11/593,312, filed on Nov. 3, 2006, and later issuedas U.S. Pat. No. 7,835,000. The foregoing applications and patents areincorporated by reference herein in their entireties for any and allpurposes.

STATEMENT OF GOVERNMENT RIGHTS

This invention was made with government support under Contract No.DE-AC52-06NA25396 awarded by the U.S. Department of Energy. Thegovernment has certain rights in the invention.

TECHNICAL FIELD

Embodiments are generally related to sensor methods and systems.Embodiments are also related to flow sensor analyzers, such as flowcytometers that move particles in a flowing fluid through a sensingregion where multiple independent optical measurements are made on theparticle. Embodiments are also related to portable systems forinterrogating particles in sample streams of flow cytometers or thelike.

BACKGROUND

Flow cytometry is a technology in which multiple physical and opticalcharacteristics of single small or microscopic particles, such as cellsor microspheres, are analyzed as they flow in a fluid stream through oneor more beams of light. Flow cytometry is an integral technology innearly every bio-medical discipline including diverse biological assaysin clinical settings. Additionally, flow cytometry is an importantanalytical platform to perform biological point detection,bio-surveillance, and forensic analysis in support of homeland defense.

In the flow cytometer, particles are carried to the light beam interceptin a fluid stream. When particles pass through the light beam, theyscatter the light and any fluorescent molecules present on or in theparticle fluoresce. These resulting optical signals are directed bymeans of optics to appropriate detectors which generate electronicsignals proportional to the optical impulses striking them. Theseelectronic signals are processed to gather data on each particle orevent and subsequently analyzed to provide information about the sample.Various particle properties, such as particle size, granularity andfluorescence intensity, can be determined by a flow cytometer recordinghow the particle under interrogation scatters the incident light beamand emits fluorescence.

Flow cytometers typically incorporate expensive lasers with highlystable outputs in order to obtain the high detection sensitivity andresolution necessary in many applications. Unfortunately, the size andexpense of typical flow cytometers currently restricts their use toclinical and laboratory environments. Such flow cytometers cost morethan 30,000 US dollars to purchase. For many potential users of flowcytometers, instrumentation size and cost are important considerationsthat may limit the acceptance of these systems in broader applications.

There is a continuing need to provide improved systems and methods formeasuring particles in a sample stream of a flow cytometer or other flowbased analyzers which can be implemented at low cost and with reducedinfrastructure requirements, such as electrical power or otherlaboratory-based requirements. Reducing size and cost nearly alwaysspeeds acceptance and adoption of new technology.

SUMMARY

The following summary of the invention is provided to facilitate anunderstanding of some of the innovative features unique to the presentinvention and is not intended to be a full description. A fullappreciation of the various aspects of the invention can be gained bytaking the entire specification, claims, drawings, and abstract as awhole.

It is, therefore, one aspect of the present invention to provide forimproved sensor methods and systems.

It is another aspect of the present invention to provide for improvedmethods and systems for measuring particles in a flow stream of a flowcytometer or like analyzer.

It is a further aspect of the invention to provide low cost methods andsystems for measuring particles in a flow stream of a flow cytometer orother flow based analyzer.

The aforementioned aspects of the invention and other objectives andadvantages can now be achieved as described herein.

According to one aspect, a system for interrogating a particle in asample stream of a flow cytometer or the like has a laser light sourcefor generating a low power light beam and a fluidics apparatus fortransporting the particle in the sample stream at substantially lowvelocity resulting in extended transit times through the light beam forinterrogation thereof Also included in the system are one or moredetectors for detecting optical signals resulting from the light beamimpinging on the particle and signal conditioning circuitry, operablycoupled to the detector(s), for conditioning output signals from thedetector(s) into electronic signals for processing thereof The signalconditioning circuitry can be a low pass filter circuitry for filteringhigh frequency noise from the detected optical signals.

Advantageously, the fluidics apparatus transports the particle in thesample stream at substantially low velocity through the focused laserbeam resulting in extended transit times to increase sensitivity and thesignal conditioning circuitry is configured to low pass filter theresulting detected optical signals, which permits high-sensitivitymeasurements to be made with simplified circuitry and low-costcomponents, such as a laser pointer and miniature detectors.

The light source can be for example a low power laser pointer modulesuch as a diode pumped solid state (DPSS) green laser. The transit timeof the particle through the light beam can be of the order of 100microseconds or more. The low pass filter circuitry can have a maximumcut off frequency of about 10 KHz.

The detector can be for example a Photomultiplier tube (PMT),photodiode, APD or hybrid detector. The signal conditioning circuitrycan comprise a pre-amplifier stage coupled to the output of a PMTdetector with the low pass filter circuitry integrated in thepre-amplifier stage. The pre-amplifier can be for example a high inputimpedance voltage follower circuit coupled to a limited band widthinverting amplifier.

The fluidics apparatus can include a hydrodynamically focused flowchamber, an acoustically focused flow chamber or an unfocused flowchamber. The flow chamber can be coupled to a slow flow delivery systemfor transporting the particle through the light beam with thesubstantially low velocity resulting in extended transit times (>100microseconds).

According to another aspect, a system for interrogating a particle in asample stream of a flow cytometer or the like has a low powered laserpointer for generating a light beam and a fluidics apparatus fortransporting the particle in the sample stream at substantially lowvelocity through the light beam for interrogation thereof Also, thesystem includes one or more detectors for detecting optical signalsresulting from the light beam impinging on the particle and signalconditioning circuitry, operably coupled to the detector(s), forconditioning output signals from the detector(s) into electronic signalsfor processing thereof The signal conditioning circuitry can include lowpass filter circuitry for filtering high frequency noise from thedetected signals.

According to another aspect, a method for analyzing a particle in asample stream of a flow cytometer or the like comprises generating a lowpower light beam; transporting the particle at substantially lowvelocity in the sample stream through the light beam for interrogationthereof, detecting light signals generated in response to the light beamimpinging on the particle, and signal conditioning the detected lightsignals into electronic signals for processing thereof, the step ofsignal conditioning comprising filtering high frequency noise from thedetected light signals.

By transporting the particle at substantially low velocity through thelight beam to increase sensitivity and low passing filtering theresulting detected optical signals, high detection sensitivity andresolution can be achieved using lower power and less stable light beamsthan those typically used in the flow cytometers of the prior art. Thesystem permits the use of low cost and compact lasers whilst providingthe detection sensitivity and resolution demanded by many applications.

The transit time of transporting the particle through the low powerlight beam can be about 100 microseconds or more. The high frequencyfiltering has a maximum cut off frequency of about 10 KHz. The low powerlight beam can be generated by a laser pointer module. The low powerlight beam can be detected by a PMT. The step of transporting theparticle can include hydro-dynamically or acoustically focusing thesample stream through a flow chamber.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying figures, in which like reference numerals refer toidentical or functionally-similar elements throughout the separate viewsand which are incorporated in and form a part of the specification,further illustrate the present invention and, together with the detaileddescription of the invention, serve to explain the principles of thepresent invention.

FIG. 1 illustrates a block diagram of a system for interrogating amicroscopic particle in a sample stream of a flow cytometer according toa preferred embodiment;

FIG. 2 illustrates the fluidic apparatus of FIG. 1 in more detail;

FIG. 3 illustrates a cross-sectional view of the flow cell taken alongline A-A′ of FIG. 2;

FIG. 4 illustrates a schematic circuit diagram of a high input impedancelimited bandwidth pre-amplifier of the system depicted in FIG. 1;

FIG. 5 illustrates a flow diagram outlining a method for interrogating aparticle in a sample stream of a flow cytometer according to a preferredembodiment;

FIG. 6 illustrates a histogram of the Side Scatter (SSC) amplitudeparameter on 10000 simulated events that demonstrates the stability ofthe laser pointer by virtue of the 1.24% coefficient of variation (CV)of the distribution;

FIG. 7 demonstrates the stability of the laser pointer by displaying SSCpeak (amplitude) versus time during the entire 4 minutes required tocollect 10000 simulated events;

FIG. 8 illustrates high-resolution and high-sensitivity performance on 6populations of microspheres in the fluorescence area histogram, obtainedby analyzing a mix of 1.87 and 2.8 μm blank (i.e. non-fluorescent)microspheres added to the RCP-20-5 2.1 μm diameter microsphere set usingthe system of FIG. 1;

FIG. 9 illustrates high-resolution SSC performance in a contour plot ofSSC peak Vs fluorescence area, obtained by analyzing a mix of 1.87 and2.8 μm blank microspheres added to the RCP-20-5 2.1 μm diametermicrosphere set using the system of FIG. 1;

FIG. 10 displays a contour plot of SSC Peak Vs SSC Width obtained byanalyzing the RCP-30-5A microsphere mixture with the system of FIG. 1,which illustrates the region of interest around the main peak used togate the fluorescence data in FIGS. 11 and 12.

FIG. 11 illustrates excellent resolution of all 8 microspherepopulations (in the 16-bit fluorescence peak data) obtained by analyzingthe RCP-30-5A microspheres sample using the system of FIG. 1;

FIG. 12 illustrates excellent resolution of all 8 microspherepopulations (in the 32-bit area data) obtained by analyzing theRCP-30-5A microspheres sample using the system of FIG. 1; and

FIG. 13 demonstrates the linearity of the system in a graph offluorescence area Vs the calibrated intensity of the RCP-30-5Amicrospheres (in units of Mean Equivalent Soluble Fluorophore forphycoerythrin MESF-PE) obtained by measuring the RCP-30-5A microspheresusing the system of FIG. 1.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

The illustrative embodiment provides an approach to interrogatingmicroscopic particles in a sample stream of a flow cytometer, or othersystems that use the flow cytometry paradigm, using a method and asystem which enables a compact and inexpensive flow cytometer to beimplemented, while having high detection sensitivity and resolutioncomparable to that of prior art flow cytometers.

Referring to FIGS. 1 and 2 of the accompanying drawings, which,respectively, illustrate block diagrams of the optical-electricalcircuitry and fluidic circuitry of the system for measuring particles ina sample stream of a flow cytometer according to one embodiment. Thesystem 1 has a light source 2 for generating a low powered light beam 3and a fluidics apparatus 26, which is configured to transport particlesin the sample stream 30 at a substantially low velocity through thelight beam for interrogation. Detectors 4, 5 are configured to detectoptical signals generated in response to the light beam 3 impinging theparticles. Signal conditioning circuitry 6, 7 can be connected to eachof the detectors 4, 5 to condition each detector output 81A, 81B intoelectronic signals 82A, 82B for processing and can be designed togenerate a limited frequency response in order to filter high frequencynoise from the detector output signals.

As will be explained in more detail below, configuring the system totransport particles in the sample stream at substantially low velocitythrough the light beam and filtering high frequency noise from thedetector output using limited bandwidth signal conditioning circuitry,permits a compact and inexpensive diode pumped solid state laser to beused in a flow cytometer while maintaining high detection sensitivityand resolution using miniature detectors that require minimal supportcircuitry.

In the illustrative embodiment of the system of FIG. 1, the light source2 for generating the low powered light beam is a low powered laser,typically having a 10 mW or less output. For example, the light sourcecan be an inexpensive diode pumped solid state (DPSS) laser, such as alaser-pointer module. One example of a suitable laser pointer is an OEMversion of a commercially available laser pointer (532 nm, 3.0 mW, modelGMP-532-5F3-CP) from LaserMate Group, Inc. which emits a green laserbeam. The laser pointer operates on 2.1-3.0 VDC and has a rated outputof 3-5 mW. A power source 40, such as Tektronix power supply (modelPS281), provides the DC voltage (typically 2.3 VDC @270 ma).Alternatively, a battery or very simple line-voltage operated powersupply can be utilized to power the laser pointer with no observabledegradation of the system performance. The laser head itself is a verycompact device with reasonable manufacturer's specifications (TEM00,<1.4 mrad beam divergence, M² <2, and 5% output power stability).Advantageously, the laser pointer module is inexpensive, compact andresults in the entire excitation source using less than 1 W of power,which greatly increases instrument portability.

Optics 9-11 are configured to direct the laser beam 3 to the analysisregion 35 of the system, that is, the point where the stream 30, whichis flowing from out of the page of FIG. 1, intercepts the light beam 3.A half-wave plate 9 and polarizing beam splitter 10 positioned betweenthe light source 2 and a mirror 11, serve to attenuate the laser beam(to the desired power level). Mirror 11 is configured to reflect thebeam 3 onto a focusing lens 12, which focuses the beam to a high lightflux (10 μm diameter spot) at the analytical region 35 of the system.

Detectors 4, 5, which are configured as side-scatter light (SSC) andfluorescence (FL) detectors, respectively, are aimed at the analyticalregion 35 of the system. Collection lenses 13, 14 are arranged on eitherside of the flow cell 25 to collect and concentrate light onto thedetectors 4, 5 through respective filters 15 (SSC), 16 (FL).

In the illustrative embodiment of the system of FIG. 1, the detectors 4,5 are photomultiplier tubes which detect the optical impulse generatedwhen the particle passes through the light beam 3 and produces a currentsignal proportional to the intensity and duration of the impulse. Thephotomultiplier tubes utilized in this example are miniature Hamamatsu5783 or 6780 Photomultiplier tubes. These multi-alkali metal-packagedetectors typically have a gain of about 10⁶ and radiant sensitivity of60-80 mA/W in the 400-700 nm range, depending on the specific modelused, and are operable with a high voltage of 250 to 1000V The exactvoltage utilized depends on the individual PMT, the laser output power,and the intensity of the signals being measured (fluorescence or lightscatter), which in turn is related to the particles being analyzed.Other low cost and compact light detectors can alternatively beemployed. The detectors could be photodiodes, avalanche photodiodes orany small detector capable of detecting light emission. Utilizing lowcost, compact PMTs further reduces the cost and size of the system.

Signal conditioning circuitry 6, 7 consists of pre-amplifiers connectedto the anode of PMT detectors 4, 5 to provide high input impedance andlimited bandwidth. The primary function of a pre-amplifier is to converta current signal to a voltage signal for further use by data acquisitionelectronics. A power supply module 19 is assembled and electricallycoupled to the detectors 4, 5 and signal conditioning circuitry 6, 7 toprovide typically + and −5 VDC (200 ma) for the pre-amplifiers 6, 7, and+15 VDC (200 ma) to power and control the high-voltage 17, 18 for eachPMT 4, 5. Further signal amplification, if needed, can be provided in adata acquisition system 80 which is coupled to the signal conditioningcircuitry outputs for collection of the conditioned PMT output signals82A, 82B.

Signal conditioning circuitry 6, 7 is designed to have limited bandwidthto filter any high-frequency noise in the PMT detector outputs. As shownin FIG. 4, which is a schematic circuit diagram of an example of thepre-amplifier circuitry, the pre-amplifier circuitry has a high inputimpedance voltage follower 60 coupled to a limited bandwidth invertingamplifier 61 in each of the pre-amplifiers 6, 7. The inverting amplifier61 has an effective bandwidth of approximately 10 kHz and amplificationfactor of 3.3. Voltage follower 60 has a pair of resistors 62, 63serially connected between the non-inverting input 65 of an operationalamplifier 50 and ground 70 and provides high impedance to the output 71of a respective detector 4, 5 which output is connected across resistor63. The voltage follower output 72 is connected via input resistor 66 tothe inverting input 69 of an operational amplifier 51 of the amplifier61. A resistor 68 and capacitor 67 (RC) circuit is arranged in thefeedback path of the operational amplifier between the operationalamplifier output and the inverting input 69 and is selected to have anRC constant to provide a maximum cut off frequency of about 10 kHz.

Low pass filters other than the limited-bandwidth amplifier 61 can beutilized in system 1 to achieve the desired high-frequency filtering andneed not necessarily be integrated in the pre-amplifier circuit. Forexample, the low pass filter could alternatively be implemented insignal conditioning circuitry after the pre-amplifier.

Incorporating a limited bandwidth pre-amplifier (effectively a low-passfilter) 61 in the signal conditioning circuitry is advantageous in thatthe high-frequency noise produced by low cost, relatively unstable lightsources, such as laser pointer module, can be filtered out from thedetected optical signals. By extending the transit times of theparticles through the light beam to increase sensitivity and low passingfiltering the resulting detected optical signals, high detectionsensitivity and resolution can be achieved using lower power and lessstable light beams than those typical utilized in the flow cytometers ofthe prior art. The system permits the use of low cost and compact laserswhilst providing the detection sensitivity and resolution demanded bymany applications. Slow flow, that generates extended transit times ofthe particles through the focused light beam, permits high-sensitivitymeasurements to be made with simplified circuitry and low-costcomponents, such as the laser pointer and miniature detectors, to createa portable battery powered system with high performance. Both of thesecomponents have minimal power requirements which makes it possible tooperate the system off of a battery or simple power supply such as a“wall wart” and 3-terminal voltage regulators with resulting performanceis at least as good as the state-of-the-art systems currently available.

In typical flow cytometry data processing the pulse (or impulse) causedby a particle passing through the laser beam is characterized by 3measurements: amplitude (or peak), duration, and area, and it is thiscorrelated data collected by the data acquisition system that can beplotted as 1D histograms (as in FIG. 6). The correlated data can also bedisplayed in 2D as dot plots or contour plots (as in FIG. 9), or otherdisplays depending on the software utilized. Specific regions on theseplots can be sequentially separated by a series of subset extractionswhich are termed gates. Specific gating protocols exist for diagnosticand clinical purposes. The plots are often made on logarithmic scales.

A data acquisition system 80 is configured to collect conventional flowcytometric data files in which the event-based parameters of height,width, and area were collected from the electronic pulses derived fromdetectors 4, 5 and pre-amps 6, 7. Custom hardware boards are configuredto convert the pre-amp output (2 V peak-to-peak) into a 14-bit digitaldata stream using a free running 14-bit 40 MS/sec ADC (AnalogDevices—ADS5421Y). The output from each pre-amp 6, 7 is connecteddirectly to separate ADC inputs. A field programmable gate array on thecustom board captures the correlated digitized waveforms and sends themto a commercial digital signal processor board (OrSys—microlineC6211CPU). The digital signal processor (Texas Instruments—TMS320C6211)extracts the pulse height, pulse area and pulse width parameters,sending the list-mode results to the host computer over FireWire(IEEE1394). The pulse parameters are recorded in FCS 3.0 data files with24 bits for area, 16 bits for peak, 12 bits for width, and 28 bits fortime (1 msec resolution). The pulse height, area and width were recordedin Flow Cytometry Standard (FCS) v.3.0 data files, which is an industrystandard data file format for flow cytometry. Data acquisitiontechniques for flow cytometry are known in the art and will not bedescribed in any more detail here.

Referring now in more detail to the fluidic apparatus 26 of FIG. 2, thefluidic apparatus includes a flow chamber 25, which in this particularembodiment is an optical flow cell 25, and a slow-flow sample deliverysystem 37 configured to pass the sample through the flow cell 25 atsubstantially low velocity (˜5 cm/sec) such that the particles haveextended transit times across the beam 3. Typically, these extendedtransit times are about 100 microseconds or more. Advantageously, by thefluidic apparatus extending the particles' transit times across the beam3, the particle under interrogation has a longer residence time in theanalytical region 35 thereby increasing the system detection sensitivityand resolution for a given light beam power so that relativelyinexpensive miniature PMTs provide adequate performance.

The slow-flow sample delivery system 37 has gravity driven sheath flowfrom a sheath bottle 27, which is suspended above the flow cell 25 suchthat sheath fluid is fed to the flow cell via a sheath delivery line 28connected between the lower end of the bottle and a side port of theflow cell assembly. A sample delivery tube or capillary 31 is connectedto the bottom end of the flow cell 25 such that the sample to beinterrogated can be delivered under pressure to the flow cell. In thecenter of the flow cell is a 250×250 nm square flow channel 30 and thesample delivery tube 31 occludes about 75% of the square flow channel,as best shown in FIG. 3, which illustrates a cross-sectional view of theflow cell taken along line A-A′ of FIG. 2. The capillary 31 has a 40 nminner diameter ID 31A as indicated by the white circle in FIG. 3. Thecapillary, with an outer diameter of 245 nm, is inserted about 5 mm intothe square flow channel 30, and this occlusion results in relatively lowvolumetric flow rates of sheath for focusing of particles. The lowsheath rate results in extended transit times through the laser beam. Awaste line 32 couples the upper end of the flow channel (of the flowcell) to a waste reservoir 33 that has a fluidic head to preventoscillation in fluid flow rates. The waste line is connected to the flowcell upper end (cuvette) 39 by a short length of soft silastic rubbertubing 41 that is gently pressed up to the top of the cuvette and heldin place by a metal arm. By adjusting the relative heights of the sheathsupply bottle 27 and the waste reservoir 33, the transit time of singleparticles across the laser beam 3 can be varied from about 100microseconds to milliseconds. The direction of flow in the flow cell isfrom bottom to top to help clear bubbles, as show in FIG. 2, and flowingout of the page in FIG. 1. The sample is introduced through the sampletube 31 and into flow cell 25 for focusing via the sheath stream. Thesample is pushed in via pressure, while the sheath is gravity fed in thecorners of the flow channel around the capillary. The sheath bottle canbe raised above the flow cell (for gravity delivery) and pressurized toset the transit time.

In the illustrative embodiment of the system of FIG. 1, the flow cell 25is a hydrodynamically focused flow cell. However, any particle focusingtechnique (e.g. acoustic, dielectrophoretic, etc . . . ) that results inextended transit times (100 microseconds to milliseconds) can beemployed. Furthermore, any flow chamber suitable for passing the samplestream through the light beam could be employed. All that is required isthat the particle passes (centered in the flow channel) through thelight beam 3 with sufficiently slow velocity so that the low poweredlight beam striking the particle results in optical signals which aredetectable with sufficiently high sensitivity to enable the use of lowpowered laser beams.

A method 100 for interrogating a particle in a sample stream of a flowcytometer according to one embodiment will now be described withreference to FIG. 5 which is a flow diagram outlining the steps of themethod 100. The method 100 can, for example, be implemented in thesystem of FIG. 1. As a general overview, initially a light beam isgenerated as indicated in step 101 of FIG. 5. In the system of FIG. 1,the light beam 3 is a low power light beam generated from a laser powermodule. The particle is transported in a sample stream through thegenerated light beam at substantially low velocity (step 102). Forexample, this step can be implemented in the system of FIG. 1 byhydro-dynamically or acoustically focusing the sample stream through theflow cell 25 and delivering the sample using the slow flow deliverysystem 37 to transport the particle through the laser beam 3 with avelocity which is sufficiently low to extend the transit time ofparticle through the light beam to about 100 microseconds or more.

Thereafter, optical signals, generated in response to the light beamimpinging on the particle, are detected (step 103 of FIG. 5). Thislatter step can be performed by the PMT detectors 4, 5 of the system ofFIG. 1. High frequency noise is then filtered from the detected lightsignals (step 104) for processing (step 105). In the system of FIG. 1,step 104 is implemented by means of the limited bandwidth invertingamplifier 61 of the signal conditioning pre-amp and step 105 isperformed by the data acquisition system of the system of FIG. 1.

Experimental Examples and Results

Specific results that have been obtained using the system and method ofinterrogating a particle in a sample stream of a flow cytometeraccording to the illustrative embodiments will now be described in whichthe interrogated samples were fluorescent calibration microsphere sets.The microsphere samples were concentrated 5-10× via centrifugation priorto use to compensate for the volumetric flow rate in this example(˜0.3-0.6 μl/minute) due to the narrow bore (40 μm ID) quartz capillarytubing utilized to deliver the sample to the flow cell. All microspheresamples were purchased from Spherotech Inc. (Libertyville, Ill.), andincluded: Rainbow Calibration Particles RCP-30-5A (8 peaks), RCP-20-5 (4peaks), CP-15-10 (blank 1.87 μm dia.), and CP-25-10 (blank 2.8 μm dia.).The specific type used is given in the text for each example.

In these examples, the laser 2 was the aforementioned green laserpointer module (532 nm, 3.0 mW, model GMP-532-5F3-CP) from LaserMateGroup. The miniature Hamamatsu PMTs described above were employed asdetectors 4, 5, which were mounted in optical tubes that held bandpassfilters 14, 15 and the light collection lenses 12, 13 in a light-tightassembly surrounding the flow cell 25. The high NA aspheric lenses 12,13 collected light from the flow cell, which was passed through bandpass filters to the detectors 4, 5. A 515-545 nm band pass filter wasused in the side-scatter (SSC) light channel while a 565-605 nm bandpass filter was used in the fluorescence channel.

In these examples, high purity water served as the sheath fluid whichwas gravity fed from a 2-L sheath bottle 27 suspended about 20 cm abovethe flow cell 25, which was a 2 cm long fused-silica cuvette 2.5×2.5 mmcross-section with a 250×250 μm flow channel. Polyethylene tubing (0.75mm ID, about 1.5-m long) was utilized as the waste line 32. A 3-m longcontinuous column of water, from the sheath bottle 27 to the wastereservoir 33, stabilized the slow flow through the gravity-drivensystem. Sample solutions were driven from a 0.5-ml Eppendorf tube bynitrogen gas pressure through the sample delivery tube 31 which was aPolymicro Technologies (Phoenix, Ariz.;) fused silica capillary tube (40μm ID, 245 μm outer diameter (OD), ˜30-cm long) inserted 4-5 mm into the250 μm square channel of the flow cell. Sheath fluid flowed around thecapillary, in the corners of the channel, vertically (from bottom to topin FIG. 2; out of the page, in FIG. 1), which facilitated dislodgingbubbles from the flow cell. The inserted tip of the capillary, ground toa 14° taper by New Objective, Inc. (Cambridge, Mass.), is positioned 300μm below the interrogation region 35. The small-bore capillary servedtwo purposes. First, it predisposed the fluidic system to slow-flowbecause it occluded about 75% of the flow channel. Second, its flowresistance permitted control of the sample delivery rate with asensitive electronic pressure regulator (MiniPR-NC-1500-5-NR; RedwoodMicrosystems, Menlo Park, Calif.). The elevated sheath bottle 27 waspressurized to provide ˜2 psig in order to reach a transit time of about250 microseconds.

The combination of scattered and fluorescent light was collected by thecollection lenses 12, 13, filtered by filters 14, 15 and picked up bythe detectors 4 (SSC) 5 (Fluorescence), and fluctuations in brightnessat each detector were converted to electrical signals which wereconditioned by the signal conditioning circuitry, analogue to digitalconverted and then processed into data. FCS files were analyzed usingFCS Express from DeNovo Software.

Laser stability was first measured by slightly misaligning the flow cellso that some of the laser light was refracted into the SSC detectorpathway (without any particles in the flow channel). The dataacquisition system 80 was electronically triggered to collect 10000simulated events, recording the pulse height, width and areameasurements from the SSC channel. These results, depicted in FIG. 6,indicate that the laser output was stable during the data collectioninterval and the precision of the measurements is indicated by thecoefficient of variation (CV) of the histogram peak, which was 1.24% forboth SSC Peak and SSC Area. The laser stability over time is clearlyshown by the stability of the scattered light over 4 minutes ofcollection (FIG. 7).

The pulse data for a series of microspheres were collected using thedata acquisition system 80. FIG. 8 demonstrates excellent resolution andsensitivity obtained from analyzing a mixture of the 4 population set ofRCP-20-5 microspheres with non-fluorescent 1.9 μm (CP-15-10) and 2.8 μm(CP-25-10) polystyrene microspheres added. The three dimmestmicrospheres that are not well resolved in the Fluorescence Areahistogram (below 10⁴) of FIG. 8, are completely resolved in the contourplot of FIG. 9, a 2D display of SSC versus Fluorescence Area.

The data depicted in FIGS. 10-13 resulted from analyzing the RCP-30-5Amicrosphere mix with the system depicted in FIG. 1. SSC width versus SSCarea are displayed in a contour plot to indicate the gating region usedto identify the individual microspheres (see FIG. 10), which are thenpresented in 1D histograms in FIGS. 11 and 12. FIG. 11 shows baselineresolution of all 8 microsphere populations using the amplitudeparameter. FIG. 12 displays exemplary data on the area parameter withall 8 populations baseline resolved over a span of 5 orders ofmagnitude. As shown in FIG. 13, plotting the means of the fluorescencearea peaks (the means for the 8 populations) against the estimated meanequivalent soluble fluorophore (MESF) values measured in unitsPhycoerythrin (MESF-PE) molecules, provided by the manufacturer,demonstrates the excellent linearity and sensitivity of the system.Extrapolating from this linear fit of fluorescence vs. MESF PE suggeststhat the system can detect as few as 50 fluorphores of PE per particle.

The components of the system in this example (laser pointer, PMTs andaspheric lenses for focusing and collection optics) cost approximately$1000. Using 1 mW of laser power through the system to analyze RCP-30-5Amicrospheres resulted in baseline resolution of all 8 peaks anddemonstrated detection of ˜50 fluorophores per particle. With theexception of particle analysis rate and number of parameters, thisexperimental example of the system has demonstrated comparableperformance to that of flow cytometers that use expensive lasers anddetectors that cost >$10,000 using components that cost less than $1000.

The aforementioned experimentation and results are for illustrationpurposes only and are not intended in any way to limit embodiments ofthe system or method to such an example. The system and method of theillustrative embodiments could be implemented in a range of differentapplications, such as biomedical diagnostics, homeland defense and pointof care devices, to measure other particles and particle parameters.Examples of such particles and measuring parameters are volume andmorphological complexity of cells cell pigments, DNA (cell cycleanalysis, cell kinetics, proliferation etc.), RNA, chromosome analysisand sorting (library construction, chromosome paint), proteins, cellsurface antigens (CD markers), intracellular antigens (variouscytokines, secondary mediators etc.), nuclear antigens, enzymaticactivity, pH, intracellular ionized calcium, magnesium, membranepotential, membrane fluidity, apoptosis (quantification, measurement ofDNA degradation, mitochondrial membrane potential, permeabilitychanges), cell viability, monitoring electropermeabilization of cells,oxidative burst, characterizing multi-drug resistance (MDR) in cancercells, glutathione, various combinations (DNA/surface antigens etc.).Other examples of such particles and measuring parameters are pollen,spores, paint pigment particles, plankton, and other small ormicroscopic organisms.

The embodiments and examples set forth herein are presented to bestexplain the present invention and its practical application and tothereby enable those skilled in the art to make and utilize theinvention. Those skilled in the art, however, will recognize that theforegoing description and examples have been presented for the purposeof illustration and example only.

Other variations and modifications of the present invention will beapparent to those of skill in the art, and it is the intent of theappended claims that such variations and modifications be covered. Forexample, in the illustrative embodiment of the system 1 depicted in FIG.1, a single laser is employed to provide a light beam 3 and a pair ofdetectors 4, 5 are arranged to detect side scatter and fluorescence.However, the system can have a single detector or more than twodetectors and/or more than one laser as required. Furthermore, whilstthe system of the illustrative embodiment is arranged to measure aplurality of particles passing through the light beam in succession, asis known in flow cytometer technology, the system could alternatively beconfigured to simply measure a single particle. Those skilled in the artwould also understand that the system and method for analyzing aparticle in a sample stream can be implemented in flow based analyzersother than flow cytometers.

The description as set forth is not intended to be exhaustive or tolimit the scope of the invention. Many modifications and variations arepossible in light of the above teaching without departing from the scopeof the following claims. It is contemplated that the use of the presentinvention can involve components having different characteristics. It isintended that the scope of the present invention be defined by theclaims appended hereto, giving full cognizance to equivalents in allrespects.

What is claimed:
 1. A particle interrogation system comprising: a flow chamber; a fluid container in connection with the flow chamber, the fluid container configured to deliver a gravity fed fluid to the flow chamber; a laser source configured to impinge a laser beam on one or more particles flowing in the flow chamber; a particle delivery system operably coupled to the flow chamber and configured to deliver a sample fluid containing one or more particles in the flow chamber, the particle delivery system being configured to provide a transit time of between at least about 100 microseconds to about 1 millisecond for a particle transported through the laser beam; at least one detector configured to receive one or more optical signals resulting from fluorescence or light scattered from the one or more particles; and signal conditioning circuitry, operably coupled to the at least one detector.
 2. The system of claim 1, wherein the laser source comprises a non-stabilized compact laser.
 3. The system of claim 1, wherein the fluid container is connected to the flow chamber via a gravity fed sheath delivery line.
 4. The system of claim 1, wherein the signal conditioning circuitry comprises a pre-amplifier stage coupled to the output of the detector, and wherein the low pass filter circuitry is integrated in the pre-amplifier stage.
 5. The system of claim 1, wherein the flow chamber is one or more of a hydrodynamically focused flow chamber, an acoustically focused flow chamber or an unfocused flow chamber.
 6. The system of claim 1, wherein the flow chamber is rectangular and wherein the particle delivery system comprises a cylinder, a length of the cylinder being inserted into the flow chamber.
 7. The system of claim 1, wherein the laser source has an output power of less than 10 mW.
 8. A method for interrogating one or more particles in a flow chamber, the method comprising: receiving from a fluid container a gravity based flow of a sheath fluid; flowing the sheath fluid in a flow chamber; delivering a sample fluid including one or more particles in the flow chamber; flowing, at a velocity, the one or more particles through a laser beam from a laser source impinging on the one or more particles flowing in the flow chamber under such conditions that the one or more particles has a transit time through the laser beam of between about 100 microseconds to about 1 millisecond; detecting scattered light or fluorescence resulting from the laser beam impinging on the one or more particles; and signal conditioning the scattered light or fluorescence into electronic signals for processing thereof
 9. The method of claim of claim 8, wherein signal conditioning comprises filtering high frequency noise from the detected scattered light or fluorescence.
 10. The method of claim 8, wherein the gravity based flow of fluid is received via a gravity fed sheath delivery line.
 11. The method of claim 8, wherein the scattered light or fluorescence is detected by at least one of a PMT photodiode, an avalanche photodiode, or a hybrid detector.
 12. The method of claim 8, wherein the laser beam is generated by a non-stabilized compact laser.
 13. The method of claim 8, wherein a rate of flow of the sheath fluid is determined by adjusting a relative height between the fluid container and a waste reservoir.
 14. The method of claim 8, further comprising hydrodynamically or acoustically focusing the sample fluid flowing in the flow chamber.
 15. The method of claim 8, wherein the flow chamber is rectangular and wherein the particle delivery system comprises a cylinder, a length of the cylinder being inserted into the flow chamber.
 16. The method of claim 8, further comprising adjusting the velocity of the one or more particles based on the power output of the laser source.
 17. The method of claim 8, wherein the laser source has an output power of less than 10 mW.
 18. A particle interrogation apparatus comprising: a flow chamber; a sheath fluid container attached to the flow chamber via a gravity fed sheath delivery line, the sheath fluid container configured to deliver a gravity based flow of a fluid to the flow chamber; a particle delivery system configured to deliver a sample fluid including one or more particles in the flow chamber; a laser source configured to impinge a laser beam on the one or more particles in the flow stream, the apparatus being configured to provide a transit time of between about 100 microseconds and 1 millisecond for a particle transported through the laser beam; and at least one detector configured to receive optical signals resulting from the laser source impinging on the one or more particles.
 19. The particle interrogation apparatus of claim 18, wherein the flow chamber is rectangular and wherein the particle delivery system comprises a cylinder, a length of the cylinder being inserted into the flow chamber.
 20. The particle interrogation apparatus of claim 18, wherein the laser source has an output power of less than 10 mW. 